Streamlined Workflows for N-Glycan Analysis of Biotherapeutics Using Agilent AdvanceBio Gly-X InstantPC and 2-AB Express Sample Preparation with LC/FLD/MS

Significance of the Topic

Glycosylation is a critical quality attribute in biotherapeutic development, impacting protein stability, immunogenicity and therapeutic efficacy. Comprehensive profiling of released N-glycans ensures consistent product performance and regulatory compliance.

Objectives and Study Overview

This study compares two streamlined workflows for N-glycan analysis of monoclonal antibodies and Fc-fusion proteins using Agilent AdvanceBio Gly-X kits. One workflow employs InstantPC glycosylamine labeling and the other uses traditional reductive amination with 2-aminobenzamide (2-AB). Key goals include evaluating preparation speed, chromatographic separation, fluorescence response and mass spectrometry sensitivity.

Methodology and Used Instrumentation

Released N-glycans were obtained via PNGase F deglycosylation after a three-minute denaturation at 90 °C and five-minute digestion at 50 °C. InstantPC labeling required one minute at 50 °C, while the 2-AB workflow used 60 minutes at 80 °C without prior glycan drying. Cleanup was performed by HILIC-SPE on-matrix.

Used Instrumentation:

• Agilent 1290 Infinity II UHPLC with AdvanceBio Glycan Mapping column (2.1 × 150 mm, 1.8 µm)
• Agilent 1260 Infinity II Fluorescence Detector (InstantPC Ex/Em=285/345 nm; 2-AB Ex/Em=260/430 nm)
• Agilent 6545XT AdvanceBio Q-TOF MS (dual AJS ESI, positive mode, m/z 600–3000)
• Data processed with Agilent MassHunter BioConfirm and Personal Compound Database

Main Results and Discussion

Both workflows achieved baseline separation of major N-glycan species (e.g. G0F, Man5, G1F isomers, G2F, G2S2, G2FS2) on a 60-minute HILIC gradient. Four replicate preparations of Rituxan and Enbrel showed relative standard deviations below 3% for high-abundance glycans. InstantPC labeling shortened total prep time to ~1 hour versus ~2 hours for 2-AB. Fluorescence peak areas and MS ion counts for InstantPC-labeled glycans were markedly higher than those for 2-AB, enabling confident detection of low-level species.

Benefits and Practical Applications of the Method

• High throughput: rapid glycan release and labeling within one hour
• Reproducibility: low inter-replicate variability supports QA/QC workflows
• Enhanced sensitivity: stronger fluorescence and MS signals facilitate detection of trace glycans
• Compatibility: workflows integrate seamlessly with existing UHPLC-FLD/MS systems

Future Trends and Potential Applications

Advancements may include automated sample preparation platforms, novel MS-friendly glycan tags, expansion to site-specific glycopeptide analysis and integration of AI-driven data interpretation. Emerging techniques in capillary electrophoresis and ion mobility could further increase glycan resolution and throughput.

Conclusion

Agilent AdvanceBio Gly-X kits enable streamlined, high-performance N-glycan analysis for biotherapeutics. InstantPC glycosylamine labeling offers significant gains in speed and sensitivity, while 2-AB remains a well-established standard. These workflows support robust glycosylation monitoring in research and manufacturing settings.

Reference

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